1. Field of the Invention
Foot-and-mouth disease (FMD) is economically one of the most important animal diseases of livestock worldwide. The disease is caused by the foot-and-mouth disease virus (FMDV) and is highly infectious, spreading rapidly by contact or aerosol.
Because of the highly infectious nature of FMD, countries free of the disease maintain rigid quarantine and import restrictions on animals and animal products from infected countries in order to prevent its introduction and to allow continued active participation in international trade. The disease does not occur in the U.S., Canada or Mexico, and its continued absence from North America is a priority for the U.S. livestock industry and the United States Department of Agriculture (USDA).
Inactivated whole virus vaccines are conventionally used in FMD control programs and have been largely successful in controlling the disease. Problems associated with the current vaccines, however, include a requirement for high-containment facilities to produce the virus needed for vaccine manufacture in, the occurrence antigenic variation of the virus resulting in numerous virus serotypes and subtypes, and the inability of vaccines to rapidly induce protective immunity. There is thus a strong incentive to develop more effective vaccines which will provide better protection against early stages of virus infection. This invention relates to a novel genetically engineered vaccine against FMD which provides superior protection over existing commercial vaccines.
2. Description of the Related Art
Genetically-engineered vaccines produced according to conventional recombinant procedures are well-established in the art (as described, for example, in Current Protocols in Molecular Biology. 1994. Ausubel et al., eds. J. Wiley & Sons, NY). In addition, vectors containing particular DNAs of interest are increasingly being utilized for the delivery of genetic material for expression in vivo (Ricigliano et al., U.S. Pat. No. 5,795,872). For example, adenovirus is a double-stranded, linear DNA virus approximately 36 kilobases in length, and vectors constructed from adenoviruses are being used to express genes of interest for use in gene therapy and vaccine development (Alkhatib and Briedis. 1988. J. Virol. vol. 62, pp. 2718-2727; Chang et al. 1995. Mol. Med. vol. 1, pp. 172-181; Cheng et al. 1992. J. Virol. vol. 66, pp. 6721-6727; Chengalvala et al. 1994. J. Gen. Virol. vol. 75, pp. 125-131; Eloit and Adam. 1995. J. Gen. Virol. vol. 76, pp. 1583-1589; Fueyo et al. 1996. Oncogene. vol. 12, pp. 103-110; Gahery-Segard et al. 1997. Eur. J. Immunol. vol. 27, pp. 653-659; Gonin et al. 1995. Vet. Microbiol. vol 45, pp. 393-401; Graham and Prevec. 1992. In Vaccines: New Approaches to Immunological Problems. Ellis, R. W., ed., Butterworth-Heinemann, Massachusetts, pp. 363-390; Jacobs et al. 1992. J. Virol. vol. 66, pp. 2086-2095; Karlsson et al. 1985. Proc. Natl. Acad. Sci. USA. vol. 82, pp. 158-162; Konishi et al. 1995. J. Clin. Invest. vol. 96. pp. 1125-1130; Korst et al. 1995. Am. J. Respir. Crit. Care Med. vol. 151, pp. S75-S87; Luback et al. 1989. Proc. Natl. Acad. Sci. USA. vol. 86, pp. 6763-6767; Mayr et al. 1999. Virology. vol. 263, pp. 496-506; Mayr et al. 2001. Vaccine. vol. 19, pp. 21552-2162; Muhlhauser et al. 1995. Circ. Res. vol. 77, pp. 1077-1086; Papp et al. 1997. J. Gen. Virol. vol. 78, pp. 2933-2943; Prevec et al. 1990. J. Infec. Dis. vol. 161, pp. 27-30; Prevec et al. 1989. J. Gen. Virol. vol. 70, pp. 429-434; Rosenfeld et al. 1991. Science. vol. 252, pp. 431-434; Sheppard et al. 1998. Arch. Virol. vol. 143, pp. 915-930; Smith et al. 1991. Mol. Endocrinol. vol. 5, pp. 867-878; Zabner et al. 1993. Cell. vol. 75, pp. 207-216; He et al., U.S. Pat. No. 5,922,576). Most of these systems make use of one adenovirus in particular, human adenovirus serotype 5 (Ad5).
Human Ad5-replication-defective constructs contain deletions in the E1 region resulting in virus which can only replicate in cells that have been stably transfected with the E1 region of the adenovirus genome, i.e., 293 cells (Graham et al. 1977. J. Gen. Virol. vol. 36, pp. 59-74). Likewise, many of these vectors contain a deletion in the E3 region, that results in a loss of inhibition of the MHC class I response, leading to an increase in the ability of animals infected by these viruses to develop an immune response to the expressed foreign genes (Chengalvala et al., supra). The loss of approximately 3000 bp of coding sequence in the E1 region, and approximately 2700 bp in the E3 region, when added to the estimated 105% of genome size which adenovirus can package (approximately an additional 1800 bp), gives vectors the ability to contain about 7500 bp of foreign sequence. This construct thus provides a useful model for the design of a vaccine requiring gene expression in vivo.
Type I interferons, IFN α and β or IFN α/β, are known to have antiviral activity and are the first line of host cell defense against virus infection (Vilcek and Sen. 1996. In Virology, Fields et al., eds. Lippincott-Raven Publishers, Philadelphia). Virus-infected cells are induced to express and secrete IFN α/β which binds to specific receptors on neighboring cells, priming them to a virus resistant state via a series of events leading to activation of IFN α/β stimulated genes (ISGs). The products of these genes affect viruses at different stages of their replication cycle, and different viruses are susceptible to different ISG products. Examples of ISGs that have been extensively characterized include double-stranded (ds) RNA dependent protein kinase (PKR), 2′-5′A synthetase/RNase L and Mx.
It has been demonstrated that FMDV replication is highly sensitive to IFN-α or -β and that supernatant fluids containing porcine or bovine IFN α/β inhibit FMDV replication (Chinsangaram et al. 1999. J. Virol. vol. 73, pp. 9891-9898). To study the effect of IFN-α and -β on FMDV replication more directly, IFN α/β genes from porcine kidney (PK) and embryonic bovine kidney (EBK) cells were amplified and cloned using primers with consensus sequences specific to IFN-α and -β for each species. A clone of each IFN was sequenced and expressed in Escherichia coli (Chinsangaram et al. 2001. J. Virol. vol. 75, pp. 5498-5503, see FIG. 1). At maximal induction, bovine IFN-α and -β and porcine IFN-α were expressed at similar levels while porcine IFN-β was expressed at a lower level (Chinsangaram et al., supra, 2001, see FIG. 1). When treated at pH 2.0, serially diluted, and tested for biological activity, porcine and bovine IFN-α or -β provided a similar inhibitory effect on FMDV replication in cells from homologous species, including a pig kidney cell line (IBRS2) and EBK cells (Chinsangaram et al., 2001, supra, see Table 1). As expected, the control E. coli-expressed FMDV protein 3C, had no inhibitory effect on virus replication (Chinsangaram et al., 2001, supra). Expressed porcine and bovine IFN-α or -β also had similar antiviral activity against vesicular stomatitis virus (VSV), encephalomyocarditis virus and classical swine fever virus. In addition, it was found that, with the exception of bovine IFN-β, porcine and bovine IFN-α and porcine IFN-β could also inhibit FMDV replication in cells from the other species (Chinsangaram et al., 2001, supra, see Table 1).
Since FMDV replication in cell culture is sensitive to IFN-α and -β treatment, type I IFN may be useful as an in vivo anti-FMDV agent. It acts rapidly, and its administration should provide protection against all serotypes and subtypes of FMDV, a concern because of the antigenic diversity of FMDV. However, IFNα/β protein is rapidly cleared, and clinical use thus requires multiple injections of high doses for a prolonged period of time. Constructs comprising type I IFN genes provide an alternative means to deliver IFN protein, thus allowing animals to produce IFN endogenously for a period of time. In addition, the amount of IFN delivered can be controlled by the recombinant virus dosage. Vectors, such as recombinant replication-defective human adenoviruses, containing these genes are also effective for delivery and subsequent gene expression in vivo.